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expression constructs usp7 inhibitors
![Viral infection promotes protein expression of <t>USP7</t> that up‐regulates SOCS1 protein levels at the late stage of viral infection. (a) Western blot analysis of protein levels of SOCS1 and JOSD1 in A549 cells infected with H1N1 [multiplicity of infection (MOI) = 1·0] for the indicated times. (b) Western blot analysis of exogenously expressed Myc‐SOCS1 levels in HEK293T cells transfected with Myc‐SOCS1 and then infected with vesicular stomatitis virus (VSV) (MOI = 1·0). (c) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells transfected with Myc‐SOCS1 and then infected with VSV (MOI = 1·0). (d) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and different DUBs, followed by treatment with or without cycloheximide (50 μm) for 12 hr. (e) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, control shRNAs (shCON) or shRNAs against human USP7 (shUSP7) as indicated and then infected with H1N1 (MOI = 1·0). (f) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells transfected with Myc‐SOCS1 and then treated with dimethylsulfoxide or <t>P5091</t> (100 nm) for 12 hr, followed by infection with VSV (MOI = 1·0). (g) Western blot analysis of <t>USP7</t> <t>protein</t> levels in A549 cells infected with H1N1 (MOI = 1·0) for the indicated times. (h,i) Western blot analysis of USP7 protein levels in HEK293T cells infected with Sendai virus (SeV) (MOI = 1·0) (h) or VSV (MOI = 1·0) (i). The densities of protein bands were quantified with imageJ. (j) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells infected with VSV (MOI = 1·0), SeV (MOI = 1·0) or H1N1 (MOI = 1·0) for 24 hr. (k) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells pretreated with pyrrolidine dithiocarbamate ammonium (PDTC) (nuclear factor‐κB inhibitor, 10 μm) for 1 hr and then infected with VSV (MOI = 1·0) for 24 hr. The data were shown as fold change normalized to that in uninfected control cells. (l) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, together with control shRNAs (–) or three shUSP7 as indicated. (m) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (n) Western blot analysis of SOCS1 protein levels in A549 cells co‐transfected with Flag‐USP7 (WT or inactive C223S mutant) as indicated](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1631/pmc07011631/pmc07011631__IMM-159-309-g001.jpg)
Expression Constructs Usp7 Inhibitors, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/expression+constructs+usp7+inhibitors/P5091/pmc07011631-60-2-20
Average 94 stars, based on 69 article reviews
Expression Constructs Usp7 Inhibitors, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/expression+constructs+usp7+inhibitors/P5091/pmc07011631-60-2-20
Average 94 stars, based on 69 article reviews
expression constructs usp7 inhibitors - by Bioz Stars,
2026-09
94/100 stars
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1) Product Images from "Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1"
Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1
Journal: Immunology
doi: 10.1111/imm.13147
Figure Legend Snippet: Viral infection promotes protein expression of USP7 that up‐regulates SOCS1 protein levels at the late stage of viral infection. (a) Western blot analysis of protein levels of SOCS1 and JOSD1 in A549 cells infected with H1N1 [multiplicity of infection (MOI) = 1·0] for the indicated times. (b) Western blot analysis of exogenously expressed Myc‐SOCS1 levels in HEK293T cells transfected with Myc‐SOCS1 and then infected with vesicular stomatitis virus (VSV) (MOI = 1·0). (c) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells transfected with Myc‐SOCS1 and then infected with VSV (MOI = 1·0). (d) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and different DUBs, followed by treatment with or without cycloheximide (50 μm) for 12 hr. (e) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, control shRNAs (shCON) or shRNAs against human USP7 (shUSP7) as indicated and then infected with H1N1 (MOI = 1·0). (f) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells transfected with Myc‐SOCS1 and then treated with dimethylsulfoxide or P5091 (100 nm) for 12 hr, followed by infection with VSV (MOI = 1·0). (g) Western blot analysis of USP7 protein levels in A549 cells infected with H1N1 (MOI = 1·0) for the indicated times. (h,i) Western blot analysis of USP7 protein levels in HEK293T cells infected with Sendai virus (SeV) (MOI = 1·0) (h) or VSV (MOI = 1·0) (i). The densities of protein bands were quantified with imageJ. (j) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells infected with VSV (MOI = 1·0), SeV (MOI = 1·0) or H1N1 (MOI = 1·0) for 24 hr. (k) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells pretreated with pyrrolidine dithiocarbamate ammonium (PDTC) (nuclear factor‐κB inhibitor, 10 μm) for 1 hr and then infected with VSV (MOI = 1·0) for 24 hr. The data were shown as fold change normalized to that in uninfected control cells. (l) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, together with control shRNAs (–) or three shUSP7 as indicated. (m) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (n) Western blot analysis of SOCS1 protein levels in A549 cells co‐transfected with Flag‐USP7 (WT or inactive C223S mutant) as indicated
Techniques Used: Infection, Expressing, Western Blot, Transfection, Virus, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Mutagenesis
Figure Legend Snippet: USP7 interacts with and stabilizes SOCS1 protein. (a,b) Immunoprecipitation analysis of the interaction between Flag‐USP7 and Myc‐SOCS1 in A549 cells co‐transfected with Myc‐SOCS1 and Flag‐USP7 using anti‐Myc (a) or anti‐Flag (b) antibodies for immunoprecipitation. (c) Immunoprecipitation analysis of the interaction between endogenous USP7 and SOCS1 in A549 cells using either the isotype IgG (−) or anti‐USP7 antibodies (USP7) for immunoprecipitation. (d) Immunoprecipitation analysis of the interaction between endogenous USP7 and SOCS1 in A549 cells using either the isotype IgG (−) or anti‐SOCS1 antibodies (SOCS1) for immunoprecipitation. (e) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1, together with shCON or shUSP7, followed by treatment with cycloheximide (50 μm) as indicated. (f) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1, together with shCON (−) or shUSP7, followed by treatment with dimethylsulfoxide or MG132 (10 μm) for 12 hr
Techniques Used: Immunoprecipitation, Transfection, Western Blot
Figure Legend Snippet: USP7 removes polyubiquitination of SOCS1 dependently on its deubiquitinase activity. (a) Immunoprecipitation analysis of polyubiquitination of Myc‐SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1, HA‐ub and Flag‐USP7 as indicated. (b) Immunoprecipitation analysis of polyubiquitination of Myc‐SOCS1 in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (c) Immunoprecipitation analysis of polyubiquitination of endogenous SOCS1 in HEK293T cells transfected with empty vectors (−) or Flag‐USP7. (d) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1, HA‐ub and shUSP7 as indicated. (e) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1 and Flag‐USP7 (WT or inactive C223S mutant) as indicated
Techniques Used: Activity Assay, Immunoprecipitation, Transfection, Mutagenesis
Figure Legend Snippet: USP7 regulates interferon type I (IFN‐I) ‐induced Janus kinase–signal transducer and activator of transcription (JAK–STAT1) activation through SOCS1. (a) Western blot analysis of STAT1 phosphorylation on tyrosine 701 (p‐STAT1) in HEK293T cells transfected with control shRNA (−) or three shUSP7 and then treated with IFN‐α (1000 IU/ml) for 30 min. (b) Western blot analysis of p‐STAT1 in HEK293T cells transfected with shCON or shUSP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (c) Western blot analysis of p‐STAT1 in HEK293T cells transfected with empty vectors (CON) or Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (d) Western blot analysis of p‐STAT1 in HEK293T cells transfected with Flag‐USP7 and then treated with IFN‐α (0, 1000, 3000 IU/ml) for 30 min. (e) Western blot analysis of p‐STAT1 in HEK293T cells transfected with empty vectors (−) or Flag‐USP7 (WT or inactive C223S mutant) and then treated with IFN‐α (1000 IU/ml) as indicated. (f) Western blot analysis of phosphorylated Tyk2 on tyrosine 1054/1055 (p‐Tyk2) and phosphorylated JAK1 on serine 1022/1023 (p‐JAK1) in HEK293T cells transfected with Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (g) Western blot analysis of endogenous SOCS1 protein levels in stable SOCS1‐knockdown HeLa cells (shSOCS1: 1# and 2#) or stable control‐shRNA HeLa cells (shCON). (h) Western blot analysis of p‐STAT1 levels in stable SOCS1‐knockdown HeLa cells transfected with Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) for 30 min
Techniques Used: Activation Assay, Western Blot, Phospho-proteomics, Transfection, Control, shRNA, Mutagenesis, Knockdown
Figure Legend Snippet: USP7 regulates the strength of interferon type I (IFN‐I) signaling. (a) The ISRE‐luciferase activity was measured in HEK293T cells transfected with control shRNA (−) or three shUSP7, together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (500 IU/ml) for 18 hr. (b) The ISRE‐luciferase activity was measured in HEK293T cells transfected with Flag‐USP7, together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (500 IU/ml) for 18 hr. (c) The ISRE‐luciferase activity was measured in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant), together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (200, 500 IU/ml) for 18 hr. (d) Quantitative‐PCR analysis of IFIT1, ISG15 and ISG54 mRNA levels in HEK293T cells transfected with shCON or shUSP7 and then treated with IFN‐α (1000 IU/ml) for 8 hr. (e) Quantitative‐PCR analysis of IFIT1, ISG15 and ISG54 mRNA levels in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant) and then treated with IFN‐α (1000 IU/ml) for 8 hr. NS, not significant, *P < 0·05, ***P < 0·001 (two‐tailed unpaired Student's t‐test). All data are shown as mean ± SD of three independent replicates
Techniques Used: Luciferase, Activity Assay, Transfection, Control, shRNA, Mutagenesis, Real-time Polymerase Chain Reaction, Two Tailed Test
![USP7 regulates interferon type I (IFN‐I) ‐mediated antiviral activity. (a) Fluorescence microscopy of vesicular stomatitis virus (VSV) with a green fluorescent protein gene (GFP) in HEK293T cells transfected with shCON or shUSP7 and then pretreated with or with IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP [multiplicity of infection (MOI) = 0·5] for 20 hr. Scale bars, 100 µm. (b) Quantitative PCR analysis of mRNA levels of a representative ISG (ISG15) in HEK293T cells transfected with shCON or shUSP7 and then infected with VSV (MOI = 1·0) for 20 hr. (c) Quantitative PCR analysis of VSV viral RNA in HEK293T cells transfected with empty vectors (CON) or Flag‐USP7 and then pretreated with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV (MOI = 1·0) for 20 hr. (d) Western blot analysis of H1N1‐encoded HA protein levels in HEK293T cells transfected with increasing amounts of Flag‐USP7 and then infected with H1N1 (MOI = 1·0) for 20 hr. (e) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant) and then infected with VSV‐GFP (MOI = 0·5) for 20 hr. Scale bars, 100 µm. ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD of three independent replicates (b,c)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1631/pmc07011631/pmc07011631__IMM-159-309-g006.jpg "USP7 regulates interferon type I (IFN‐I) ‐mediated antiviral activity. ...")
Figure Legend Snippet: USP7 regulates interferon type I (IFN‐I) ‐mediated antiviral activity. (a) Fluorescence microscopy of vesicular stomatitis virus (VSV) with a green fluorescent protein gene (GFP) in HEK293T cells transfected with shCON or shUSP7 and then pretreated with or with IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP [multiplicity of infection (MOI) = 0·5] for 20 hr. Scale bars, 100 µm. (b) Quantitative PCR analysis of mRNA levels of a representative ISG (ISG15) in HEK293T cells transfected with shCON or shUSP7 and then infected with VSV (MOI = 1·0) for 20 hr. (c) Quantitative PCR analysis of VSV viral RNA in HEK293T cells transfected with empty vectors (CON) or Flag‐USP7 and then pretreated with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV (MOI = 1·0) for 20 hr. (d) Western blot analysis of H1N1‐encoded HA protein levels in HEK293T cells transfected with increasing amounts of Flag‐USP7 and then infected with H1N1 (MOI = 1·0) for 20 hr. (e) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant) and then infected with VSV‐GFP (MOI = 0·5) for 20 hr. Scale bars, 100 µm. ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD of three independent replicates (b,c)
Techniques Used: Activity Assay, Fluorescence, Microscopy, Virus, Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis, Two Tailed Test
![USP7 Small‐molecule inhibitors enhance interferon type I (IFN‐I) signaling and antiviral efficacy. (a) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1 and HA‐ub and then treated with either P5091 (100 nm) or P22077 (100 nm) for 12 hr. (b) Western blot analysis of endogenous SOCS1 protein levels in A549 cells treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr. (c) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and then pretreated with MG132 (10 μm) for 12 hr, followed by treatment with P22077 (100 nm) for 12 hr. (d) Western blot analysis of p‐STAT1 in HEK293T cells pretreated with P22077 or P5091 (100 nm) for 12 hr and then treated with IFN‐α (1000 IU/ml) as indicated. (e) The ISRE‐luciferase activity was measured in HEK293T cells co‐transfected with ISRE‐luciferase and Renilla and then treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr, followed by treatment with IFN‐α (500 IU/ml) for 18 hr. (f) Quantitative PCR analysis of mRNA levels of IFIT1, ISG15 and ISG54 in HEK293T cells pretreated with dimethylsulfoxide or P22077 (100 nm) for 12 hr, and then treated with or without IFN‐α (1000 IU/ml) for 6 hr. (g) Cell viability was measured in HEK293T cells treated with either P5091 or P22077 using different concentrations as indicated. (h) Quantitative PCR analysis of vesicular stomatitis virus (VSV) viral RNA in HEK293T cells pretreated with P5091 or P22077 (20 and 100 nm) for 12 hr and then challenged with VSV [multiplicity of infection (MOI) = 1·0] for 24 hr. (i) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells pretreated with DMSO, P5091 and P22077 (100 nm) for 12 hr and then treatment with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP (MOI = 0·5) for 24 hr. Scale bars, 100 µm. *P < 0·05, **P < 0·01 and ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD. of three independent replicates (e,f,h)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1631/pmc07011631/pmc07011631__IMM-159-309-g007.jpg "USP7 Small‐molecule inhibitors enhance interferon type I (IFN‐I) signaling ...")
Figure Legend Snippet: USP7 Small‐molecule inhibitors enhance interferon type I (IFN‐I) signaling and antiviral efficacy. (a) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1 and HA‐ub and then treated with either P5091 (100 nm) or P22077 (100 nm) for 12 hr. (b) Western blot analysis of endogenous SOCS1 protein levels in A549 cells treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr. (c) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and then pretreated with MG132 (10 μm) for 12 hr, followed by treatment with P22077 (100 nm) for 12 hr. (d) Western blot analysis of p‐STAT1 in HEK293T cells pretreated with P22077 or P5091 (100 nm) for 12 hr and then treated with IFN‐α (1000 IU/ml) as indicated. (e) The ISRE‐luciferase activity was measured in HEK293T cells co‐transfected with ISRE‐luciferase and Renilla and then treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr, followed by treatment with IFN‐α (500 IU/ml) for 18 hr. (f) Quantitative PCR analysis of mRNA levels of IFIT1, ISG15 and ISG54 in HEK293T cells pretreated with dimethylsulfoxide or P22077 (100 nm) for 12 hr, and then treated with or without IFN‐α (1000 IU/ml) for 6 hr. (g) Cell viability was measured in HEK293T cells treated with either P5091 or P22077 using different concentrations as indicated. (h) Quantitative PCR analysis of vesicular stomatitis virus (VSV) viral RNA in HEK293T cells pretreated with P5091 or P22077 (20 and 100 nm) for 12 hr and then challenged with VSV [multiplicity of infection (MOI) = 1·0] for 24 hr. (i) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells pretreated with DMSO, P5091 and P22077 (100 nm) for 12 hr and then treatment with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP (MOI = 0·5) for 24 hr. Scale bars, 100 µm. *P < 0·05, **P < 0·01 and ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD. of three independent replicates (e,f,h)
Techniques Used: Immunoprecipitation, Transfection, Western Blot, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Virus, Infection, Fluorescence, Microscopy, Two Tailed Test